PMC

The figure shows a schematic representation of the “glutathione pathway.” Glutathione (GSH) is synthesized from glutamate, cysteine, and glycine by γ-glutamylcysteine synthetase and glutathione synthetase. Glutathione redox state is regulated, in part, by glutathione peroxidases, forming oxidized glutathione (GSSG), and by a reaction catalyzed by glutathione reductase. Glutathione is conjugated to substrates both through the action of the glutathione S-transferases and through non-enzymatic reactions. Glutathione conjugates can be excreted from the cells by members of the ABCC transporter family.

LvGPX3 domain distribution and phylogenetic analysis of the GPXs. (A) Schematic representation of the structural motifs of LvGPX3; (B) Phylogenetic tree of PGXs. HsGPX, Homo sapiens glutathione peroxidase (GenBank accession No. BAA00525.1); PtGPX3, Pan troglodytes glutathione peroxidase 3 precursor (GenBank accession No. NP_001108629.1); OoGPX3, Orcinus orca glutathione peroxidase 3 (GenBank accession No. XP_004280386.2); OaGPX3, Ovis aries glutathione peroxidase 3 (GenBank accession No. XP_014951639.1); CmGPX3, Chelonia mydas glutathione peroxidase 3 isoform X2 (GenBank accession No. XP_027689463.1); GgGPX3, Gallus gallus glutathione peroxidase 3 precursor (GenBank accession No. NP_001156704.1); XlGPX3, Xenopus laevis glutathione peroxidase 3 homeolog precursor (GenBank accession No. NP_001085319.2); PmGPX3, Petromyzon marinus glutathione peroxidase 3-like (GenBank accession No. XP_032814057.1); TcGPX, Tribolium castaneum glutathione peroxidase (GenBank accession No. NP_001164309.1); ApGPX, Acanthaster planci glutathione peroxidase-like (GenBank accession No. XP_022106637.1); NlGPX3, Nilaparvata lugens glutathione peroxidase 3-like (GenBank accession No. XP_022184461.1); ZnGPX6, Zootermopsis nevadensis glutathione peroxidase 6-like isoform X2 (GenBank accession No. XP_021936526.1); LpGPX, Limulus polyphemus glutathione peroxidase-like isoform X1 (GenBank accession No. XP_013772703.1); MeGPX, Metapenaeus ensis glutathione peroxidase (GenBank accession No. ACB42236.1); PcGPX, Pomacea canaliculata glutathione peroxidase-like (GenBank accession No. XP_025090868.1); PtrGPX6, Portunus trituberculatus Glutathione peroxidase 6 (GenBank accession No. MPC16807.1); PmGPX, Penaeus monodon selenium-dependent glutathione peroxidase (GenBank accession No. AQW41378.1); LvGPXL, Penaeus vannamei glutathione peroxidase-like (GenBank accession No. XP_027224546.1).

(A) Schematic illustration of the time course for glutathione depletion and (U)¹³C glutamine tracing in primary hepatocyte experiments.
(C–G) Primary hepatocytes were isolated from 12-week-old WT mice.
(H–L) Primary hepatocytes isolated from 17- to 20-week-old WT and MPC LivKO mice.
(B) Schematic demonstrating metabolic path U-¹³C glutamine incorporation into glutathione or the TCA cycle. Green denotes ¹³C label, and gray denotes ¹²C non-label.
(C) Total glutathione determined enzymatically in primary hepatocytes following glutathione depletion induced by 40 min of treatment with 50 μM monochlorobimane; n = 3 biological replicates.
(D) Total glutathione determined after 1 h of (U)¹³C glutamine chase following glutathione depletion as in (A). During the chase period, primary hepatocytes were incubated with 5 mM (U)¹³C glutamine, 5 mM glucose, and DMSO (Veh) or 5 μM UK5099 (UK); n = 3 biological replicates.
(E) Glutathione mass isotopologue distribution after chase period described in (A); n = 3.
(F) Concentration of M+3 and M+5 glutathione calculated by multiplying total glutathione shown in (C) with the isotopologue fractional enrichment shown in (D); n = 3 biological replicates.
(G) GSH to GSSG ratios in DMSO (Veh)- and UK5099 (UK)-treated hepatocytes. Ratios are calculated from metabolite mass spectrometry signal intensity and normalized to vehicle.
(H) Total glutathione determined enzymatically in WT and MPC LivKO primary hepatocytes following glutathione depletion as in (A); n = 4 biological replicates.
(I) Total glutathione determined in WT and MPC LivKO primary hepatocytes after 1 h of (U)¹³C glutamine chase following glutathione depletion as in (A). Primary hepatocytes were incubated with 5 mM (U)¹³C glutamine, 5 mM glucose; n = 3 biological replicates.
(J) Glutathione mass isotopologue distribution after chase period described in (A); n = 4.
(K) Concentration of M+3 and M+5 glutathione calculated by multiplying total glutathione shown in (I) with the isotopologue fractional enrichment shown in (J); n = 3 biological replicates.
(L) GSH to GSSG ratios in WT and MPC LivKO hepatocytes. Ratios are calculated from metabolite mass spectrometry signal intensity and normalized to WT; n = 4 biological replicates.
Data are presented as mean ± SEM, compared by t test performed unless otherwise noted (*p < 0.05, **p < 0.01, ***p < 0.001). See also .

Mesencephalic cultures were preloaded for 24hr with either the monoethyl ester of glutathione (A) or with reduced glutathione (B).(C) Some cultures had glutathione partially depleted prior to loading by treatment for 24hr with buthionine sulfoximine (BSO, 1μM). GEE loading was carried out in the presence or absence of BSO. Following loading, cultures were washed, intracellular content extracted and glutathione quantified by HPLC. The n is from 4–5 separate experiments run in duplicate. *Different from zero GEE addition.